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PubMed1999

Self-association and domains of interactions of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by analytical ultracentrifugation and NMR experiments in trifluoroethanol/H(2)O mixtures.

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.

Amino Acid Sequence↗
PubMed1999

HIV-1 integrase: structural organization, conformational changes, and catalysis.

Integrase comprises three domains capable of folding independently and whose three-dimensional structures are known. However, the manner in which the N-terminal, catalytic core, and C-terminal domains interact in the holoenzyme remains obscure. Catalytically active recombinant IN can exist in a dynamic equilibrium of monomers, dimers, tetramers, and higher order species. Numerous studies indicate that the enzyme functions as a multimer, minimally a dimer. The IN proteins from HIV-1 and ASV have been studied most carefully with respect to the structural basis of catalysis. Although the active site of ASV IN does not undergo significant conformational changes on binding the required metal cofactor, that of HIV-1 IN does. The reversible, metal-induced conformational change in HIV-1 IN impairs the binding of some anti-HIV-1 IN monoclonal antibodies to the enzyme and results in differential susceptibility of the protein to proteolysis. This active site-mediated conformational change reorganizes the catalytic core and C-terminal domains and appears to promote an interaction that is favorable for catalysis. Other metal-dependent structural changes in HIV-1 IN include the promotion of interactions between the N terminal and the catalytic core domains and the induction of tetramers by zinc ions. The end result of these metal-induced changes is apparently the induction of an activated holoenzyme that can form a stable ternary integrase-metal-DNA complex. These structural changes, which appear to be crucial for optimum catalysis in HIV-1 IN, do not occur in ASV IN. The structural changes observed in HIV-1 IN may serve to recruit the catalytic machinery in this enzyme to a conformation that is native for ASV IN.

Animals↗
PubMed2005

Integrase inhibitors to treat HIV/AIDS.

HIV integrase is a rational target for treating HIV infection and preventing AIDS. It took approximately 12 years to develop clinically usable inhibitors of integrase, and Phase I clinical trials of integrase inhibitors have just begun. This review focuses on the molecular basis and rationale for developing integrase inhibitors. The main classes of lead compounds are also described, as well as the concept of interfacial inhibitors of protein-nucleic-acid interactions that might apply to the clinically used strand-transfer inhibitors.

Africa South of the Sahara↗
PubMed2003

New antiretroviral drugs.

Despite the availability of 16 antiretroviral drugs approved for the treatment of HIV infection, current combination regimens present challenges. Newer antiretroviral drugs are needed to improve convenience, reduce toxicity and, of particular importance, to provide antiretroviral activity against viral strains resistant to the currently available antiretroviral agents. Candidate drugs with novel properties are in development in the two currently available drug classes: HIV reverse transcriptase inhibitors (nucleoside analogs, non-nucleoside analogs and nucleotide analogs) and HIV protease inhibitors (PI). Investigational nucleoside analog reverse transcriptase inhibitors (nRTI) include emtricitabine (FTC) and amdoxovir (DAPD), and investigational non-nucleoside reverse transcriptase inhibitors (NNRTI) include DPC 083 and TMC 125. New protease inhibitors under investigation include atazanavir (BMS-232 632), tipranavir, and TMC 114. In addition, newer agents with novel mechanisms of action such as HIV entry inhibitors (that inhibit the three steps of HIV entry: CD4 attachment, chemokine receptor binding and membrane fusion) and HIV integrase inhibitors are under investigation. Investigational entry inhibitors include PRO 542 (a CD4 attachment inhibitor), Schering C (a chemokine receptor inhibitor), enfuvirtide (T-20) and T-1249, inhibitors of membrane fusion. Investigational HIV integrase inhibitors include S-1360. Continued progress in the treatment of HIV disease will result from the development of new antiretroviral drugs.

Anti-HIV Agents↗
PubMed2003

Small-molecule HIV-1 integrase inhibitors: the 2001-2002 update.

Integration of viral DNA into host cell chromosomal DNA to form a provirus is an essential step in the viral life cycle. This process is mediated by integrase (IN), a 32 KDa viral enzyme. The unique properties of IN makes it an ideal target for drug design. First, there are no cellular homologues to IN and the reactions catalyzed by IN are unique. Second, IN is absolutely required for viral replication and mutations in a number of key residues dramatically block viral replication. Third, IN has been validated as a legitimate target and the results from S-1360 (1) the only available IN inhibitor under clinical trials suggest synergistic effect with reverse transcriptase (RT) and protease (PR) inhibitors. During the past 10 years a plethora of inhibitors have been identified and some were shown to be selective against IN and block viral replication. The two most predominant classes of inhibitors have been the catechol containing hydroxylated aromatics and more recently the diketoacid containing aromatics. Herein, we review all small molecule compounds reported to inhibit recombinant HIV-1 IN with IC(50) values < 20 M during the past two years. It is important to bear in mind that the true mechanism of action and antiviral activities of many of the compounds are currently not established. However, based on the growing body of literature certain classes of compounds can be easily excluded as bona fide IN inhibitors.

Drug Design↗
PubMed2005

Design, synthesis and biological evaluation of heteroaryl diketohexenoic and diketobutanoic acids as HIV-1 integrase inhibitors endowed with antiretroviral activity.

Highly active anti-retroviral therapy (HAART) using reverse transcriptase (RT) and protease (PR) inhibitors and, more recently, inhibitors of the fusion is currently the best clinical approach in combating acquired immunodeficiency syndrome (AIDS), caused by infection from human immunodeficiency virus type 1 (HIV-1). However, this therapy does not completely eradicate the virus, so that resistant strains easily emerge. The above problem calls urgently for research on inhibitors of further viral targets such as integrase (IN), the third enzyme produced by HIV. Recently, our research group was engaged in studies on conformationally restrained cinnamoyl compounds related to curcumin as anti-IN agents. Compounds containing both a 3,4,5-trihydroxyphenyl group and a carboxylic acid function were potent IN inhibitors active against viral replication. More recently, a promising new class of inhibitors synthesized by Merck Company has emerged, which contain aryldiketoacid (ADK) functionality. The ADKs selectively inhibited the stand transfer (ST) step of integration and were proven to be effective IN inhibitors in vivo. Our interest in the field of IN inhibitors led us to design pyrrole and indole derivatives containing both a cinnamoyl moiety and a diketoacid group. A number of the cited derivatives were proven potent IN inhibitors, which selectively inhibited the ST step at submicromolar concentrations and were effective against virus replication in HIV-1 infected cells.

Anti-Retroviral Agents↗
PubMed2006

Preliminary mapping of a putative inhibitor-binding pocket for human immunodeficiency virus type 1 integrase inhibitors.

Molecular modeling studies have identified a putative human immunodeficiency virus (HIV) integrase (IN) inhibitor-binding pocket for l-chicoric acid (l-CA) and other inhibitors of IN (C. A. Sotriffer, H. Ni, and A. McCammon, J. Med. Chem. 43:4109-4117, 2000). By using site-directed mutagenesis of several amino acid residues identified by modeling studies, a common inhibitor-binding pocket on IN was confirmed for l-CA and the diketo acid L-731,988. Specifically, the single mutations E92K, Q148A, K156A, K156R, G140S, and G149S, as well as the double mutations C65S-K156N and H67D-G140A were evaluated for their effects on enzymatic activity and inhibitor susceptibility. Each recombinant IN was attenuated for 3'-end processing and strand transfer activities. Most proteins were also attenuated for disintegration; the IN that contained K156R and C65S-K156N, however, displayed disintegration activity similar to that of IN from HIV(NL4-3). All mutant IN proteins demonstrated decreased susceptibility to l-CA, while all mutant proteins except E92K and K156R demonstrated resistance to L-731,988. These data validate the computer modeling data and demonstrate that l-CA and L-731,988 share an overlapping inhibitor-binding pocket that involves amino acids Q148, C65, and H67. The resistance studies confirm that L-731,988 fills one-half of the inhibitor-binding pocket and binds to Q148 but excludes E92, while l-CA fills the entire binding groove and thus interacts with E92. These results provide "wet laboratory" evidence that molecular models of the HIV IN inhibitor-binding pocket can be used for drug discovery.

Binding Sites↗
PubMed2003

Human immunodeficiency virus type-1 integrase containing a glycine to serine mutation at position 140 is attenuated for catalysis and resistant to integrase inhibitors.

L-chicoric acid (L-CA) is a potent inhibitor of HIV integrase (IN) in vitro. In this report, the effects of a glycine to serine mutation at position 140 (G140S) on HIV IN and its effects on IN inhibitor resistance are described. HIV containing the G140S mutation showed a delay in replication. Using real-time polymerase chain reaction, the delay was secondary to a failure in integration. The mutant protein (IN(G140S)) was attenuated approximately four-fold for catalysis under equilibrium conditions compared to wild-type IN (IN(WT)) and attenuated five-fold in steady-state kinetic analysis of disintegration. Fifty percent inhibitory concentration assays were performed with IN inhibitors against both IN proteins in disintegration and strand transfer reactions. IN(G140S) was resistant to both L-CA and L-731,988, a diketoacid. HIV containing the mutation was resistant to both inhibitors as well. The G140S mutation attenuates IN activity and confers resistance to IN inhibitors, suggesting that diketoacids and L-CA interact with a similar binding site on HIV IN.

Acetoacetates↗
PubMed1999

Dissecting the role of the N-terminal domain of human immunodeficiency virus integrase by trans-complementation analysis.

The human immunodeficiency virus (HIV) integrase protein (IN) catalyzes two reactions required to integrate HIV DNA into the human genome: 3' processing of the viral DNA ends and integration. IN has three domains, the N-terminal zinc-binding domain, the catalytic core, and the C-terminal SH3 domain. Previously, it was shown that IN proteins mutated in different domains could complement each other. We now report that this does not require any overlap between the two complementing proteins; an N-terminal domain, provided in trans, can restore IN activity of a mutant lacking this domain. Only the zinc-coordinating form of the N-terminal domain can efficiently restore IN activity of an N-terminal deletion mutant. This suggests that interaction between different domains of IN is needed for functional multimerization. We find that the N-terminal domain of feline immunodeficiency virus IN can support IN activity of an N-terminal deletion mutant of HIV type 2 IN. These cross-complementation experiments indicate that the N-terminal domain contributes to the recognition of specific viral DNA ends.

Animals↗
PubMed1996

Target-sequence preferences of HIV-1 integration complexes in vitro.

Integration of reverse transcribed retroviral cDNA is not restricted to particular host DNA sequences. However, the frequency of integration into a particular phosphodiester bond is influenced by the local sequence. Here we examine the target-sequence preferences of purified HIV integrase and viral nucleoprotein complexes (preintegration complexes) isolated from freshly infected cells. We find that the three-base sequence including the integration site is not the major factor determining the frequency of integration, since identical triplets embedded in different sequences are used with very different efficiencies. However, there is a statistically significant bias against integration upstream of a pyrimidine nucleotide. The target-sequence preferences of purified integrase and preintegration complexes are very different. Strong integration sites on opposite DNA strands occur in pairs separated by five residues when preintegration complexes are used but not with purified integrase. These studies highlight the difference between the two sources of HIV integration activity and may provide the basis for a simple assay for the correct assembly of viral nucleoprotein complexes.

Binding Sites↗
PubMed1993

Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro.

The retroviral integrase (IN) protein is essential for integration of retroviral DNA into the host cell genome. To identify functional domains within the protein and to assess the importance of conserved residues, we performed site-directed mutagenesis of HIV-1 IN and analyzed the mutants in vitro for IN-mediated activities: 3' processing (att site-specific nuclease activity), strand transfer (the joining of att site oligonucleotides to target DNA), disintegration (the reverse of strand transfer), and integration site selection. Changing the conserved residue His-16 either to Cys or to Val in a proposed zinc-finger region had minimal effect on IN activities. Alteration of two highly conserved amino acid residues, Asp-116-->Ile and Glu-152-->Gly, each resulted in complete or nearly complete loss of 3' processing, strand transfer, and disintegration, whereas alteration of another conserved residue, Trp-235-->Glu, had no demonstrable effect on any of the activities in vitro. Two mutants, Asp-64-->Val and Arg-199-->Cys delta, each demonstrated differential effects on IN activities. Asp-64-->Val has no demonstrable strand transfer or disintegration activity yet maintains 3' processing activity at a diminished level. Arg-199-->Cys delta, which lacks part of the carboxyl terminus of IN, has impaired strand transfer activity without loss of disintegration activity. Use of a target site selection assay showed that all of our mutants with strand transfer activity maintain the same integration pattern as wild type IN. We conclude that not all highly conserved IN residues are essential for IN activities in vitro, zinc coordination by the proposed zinc-finger domain may not be required for the activities assayed, alteration of single residues can yield differential effects on IN activities, and target site selection into naked DNA is not necessarily altered by changes in strand transfer activity.

Alleles↗
PubMed2006

Coumarins as inhibitors of HIV reverse transcriptase.

Acquired immunodeficiency syndrome (AIDS), a degenerative disease of the immune and central nervous systems, is an enormous world-wide health threat. No cure has been found, and research is aimed at developing chemotherapy against the causative agent, human immunodeficiency virus (HIV). Chemotherapy for AIDS has progressed steadily in the past decade. However, new, effective, and less toxic chemotherapeutic agents are still needed. Plants, particularly anti-infective or immunomodulating herbal medicines, can serve as sources of new active leads to be further developed as anti-AIDS drug candidates. A lot of structurally different natural coumarins were found to display potent anti-HIV activity and continued progress is anticipated in the discovery of new leads and in the development of these agents as potential anti-AIDS drug candidates. Recent studies based on the account of various coumarins from plant sources and their analogs--synthetic coumarins, indicate that some of them serve as potent nonnucleoside RT-inhibitors, another as inhibitors of HIV-integrase or HIV-protease. The current review demonstrates the variety of coumarins of natural plant origin and synthetic coumarins having unique mechanism of action to one of the most important stage of HIV replication (RT-inhibition). The merits of selecting potential anti-HIV agents to be used in rational combination drugs design and structure-activity relationships are discussed. The scientific community is looking actively for new drugs and combinations for treatment of HIV infection effective for first-line treatment, as well as against drug-resistant mutants.

Acquired Immunodeficiency Syndrome↗
PubMed1994

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages.

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q beta, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.

Antiviral Agents↗
PubMed1995

Characterization of endonucleolytic activity of HIV-1 integrase using a fluorogenic substrate.

Retroviruses require viral DNA to be synthesized by reverse transcription in the cytoplasm followed by integration of the resulting viral DNA into the host chromosome in the nucleus. Reverse transcription and integration, essential steps in the life cycle of retroviruses, are possible targets in the development of antiviral reagents. One attractive target is the integrase protein, a product of the retroviral pol gene which is solely responsible for the retroviral integration process through cutting and joining reactions. When screening for massive numbers of antiviral agents, a rapid and precise assay is ideal. We report the application of fluorescence resonance energy transfer (FRET) with fluorescein and eosin as the energy transfer pair to characterize HIV-IN-mediated DNA cleavage reactions. Past concerns with applications of FRET to DNA were due to interactions of the fluorophore with the DNA, resulting in quenched fluorescence. However, in this study these concerns have been resolved with the use of a nucleotide analog with a 12-carbon linker arm, 5-amino (12)-2'-deoxyuridine beta-cyanoethyl phosphoramidite. Steady-state fluorescence studies show that cleavage of the fluorogenic substrate by integrase results in enhancement of quenched donor fluorescence intensity. The fluorescence assay was confirmed by autoradiographic analysis of the cleavage reaction with radiolabeled fluorogenic substrate. This fluorescence assay will facilitate both detailed kinetic studies and the rapid screening of novel integrase inhibitors.

Base Sequence↗
PubMed1996

The metal ion-induced cooperative binding of HIV-1 integrase to DNA exhibits a marked preference for Mn(II) rather than Mg(II).

In this investigation, we examine the interaction between the human immunodeficiency virus type I integrase and oligonucleotides that reflect the sequences of the extreme termini of the viral long terminal repeats (LTRs). The results of gel filtration and a detailed binding density analysis indicate that the integrase binds to the LTR as a high-order oligomer at a density equivalent to 10 +/- 0.8 integrase monomers per 21-base pair LTR. The corresponding binding isotherm displays a Hill coefficient of 2, suggesting that the binding mechanism involves the cooperative interaction between two oligomers. This interaction is quite stable, exhibiting a prolonged half-life (t1/2 approximately 13 h) in the presence of Mn2+ cations. Complexes were less stable when formed with Mg2+ (t1/2 approximately 1 h). The role of Mn2+ appears to be in the induction of the protein-protein interactions that stabilize the bound complexes. In terms of the 3'-end processing of the LTR, similar catalytic rates (kcat approximately 0.06 min-1) were obtained for the stable complex in the presence of either cation. Hence, the apparent preference observed for Mn2+ in standard in vitro integration assays can be attributed entirely to the augmentation in the DNA binding affinity of the integrase.

Base Sequence↗